The DNA of all living organisms is persistently damaged and requires continuous surveillance and repair. Unrepaired damage can lead to cancer and degenerative diseases. Most of the work published by our laboratory and others has focused upon DNA damage and repair in duplex DNA. However, many regions of human chromosomes assume unusual structures (non B-form) under physiological conditions as a consequence of their primary DNA sequence, and these structures likely contribute to the biological function of these regions. Currently, very little is known about the structural consequences of damage in these regions and how the damage is repaired. The focus of this renewal application will be on structural and potential biological consequences of DNA damage and repair in unusual DNA structures in human telomeric sequences. The ends of all human telomeres contain a repeating sequence, 5' TTAGGG, that is duplex for several thousand bases and ends with the G-rich strand for several hundred bases. Multiple structures, including loops and G-quadruplex structures form under physiological conditions, and these unusual structures interact with several proteins, forming the shelterin complex. The DNA within these regions must be able to dynamically form unusual structures to protect the ends of the chromosomes from enzymatic degradation and illegitimate chromosome fusions, but the DNA structures must also be disassembled for chromosome replication and telomere maintenance. Our preliminary data, in conjunction with published studies, indicate that some forms of DNA damage prevent the formation or relaxation of important unusual structures, and in some cases prevent the binding of critical binding proteins. Furthermore, we have observed that damaged bases within telomeric structures can create significant challenges for the base excision repair pathway. Failure to maintain telomeres can lead to telomere erosion and cellular senescence, chromosome translocations, and other potentially catastrophic biochemical events. In the four aims of this renewal application we propose to examine: 1) the impact of endogenous lesions on the capacity of telomeric sequences to form unusual structures, 2) the impact of endogenous lesions on the interaction of telomeres with telomere-binding proteins, 3) the repair of endogenous lesions within telomeric sequences, and 4) damage and repair of telomeres in mammalian cells. The proposed studies are innovative in that we are forging into a relatively unexplored area, and we are developing new experimental methods through the integration of multiple basic science disciplines. The results of the proposed studies will reveal critical aspects of chromosome maintenance, potentially explain mechanisms for some genetic alterations observed in human tumors, and provide new insights into the mechanisms of action of some currently used chemotherapy agents. PUBLIC HEALTH RELEVANCE: Human DNA is persistently damaged and must be repaired to prevent degenerative diseases and cancer. The DNA at the ends of human chromosomes is contained in structures called telomeres, and currently, very little is known about the consequences of telomere damage and how telomeres are repaired. The studies proposed here will lead to a better understanding of cancer etiology and suggest new strategies for cancer chemotherapy.